CN102260689A - Gene engineering bacteria for food-grade heterologous secretion expression of IIa-class bacteriocin, construction method and application thereof - Google Patents
Gene engineering bacteria for food-grade heterologous secretion expression of IIa-class bacteriocin, construction method and application thereof Download PDFInfo
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Abstract
The invention discloses gene engineering bacteria of food-grade heterologous secretion expression of IIa-class bacteriocin, construction method and application thereof. The invention provides gene engineering bacteria which are gene engineering bacteria obtained by introducing a DNA segment containing DNA molecules I to host bacteria; the DNA molecules I are any DNA molecules in the following molecules (1) or molecules (2) or molecules (3); the molecules (1) are the DNA molecules shown in the sequence 1 of the sequence table; the DNA molecules (2) and the molecules (1) are hybridized in strict conditions and have DNA molecules with the same function; the molecules (3) and the molecules (1) or the molecules (2) are more than 98% in homology and have DNA molecules with the same function. The gene engineering lactococcus lactis bacteria for producing IIa-class bacteriocin provided by the invention can be used for realizing over-expression, continuous production and rapid detection for IIa-class bacteriocin.
Description
Technical field
The present invention relates to genetic engineering bacterium and application thereof, particularly genetic engineering bacterium and the construction process and the application of food grade allos secreting, expressing IIa bacterioid element.
Background technology
Food very easily is subjected to microbial contamination and causes putrid and deterioratedly in processing and preserving process, adopts sanitas inhibition microorganism, and delaying corruption is one of important technology of current food fresh keeping.According to the FDA estimation, annual in the world have the food more than 20% to waste because of microbial spoilage approximately, and loss is up to 17,000,000,000 dollars.China's sanitas commonly used has kind more than 30, and year production consumes about 120,000 tons, is worth about more than 50 hundred million yuan, is a sizable industry, and the national economic development is had considerable influence.Add in the use range of state approval with 1 ‰ ratio, be equivalent to prevent 5,000 ten thousand tons of food spoilages.Yet what use in the food is Chemical Preservative mostly, influences HUMAN HEALTH, and its application more and more is subjected to the restriction of numerous countries.That the natural biological antiseptic agent has is safe, nontoxic, suitability wide, steady performance, and therefore, exploitation high-efficiency broad spectrum biological food antiseptic has become the vital task of modern food industry.
Bacteriocin is bacterium synthetic justacrine in metabolic process has bacteriostatic activity to the class in the environment polypeptide or a protein matter, it can be degraded in human body, has nontoxic, noresidue, efficiently, characteristics such as acidproof, high temperature resistant, no resistance, become the focus of natural antiseptic agent research and development at present.
II a bacterioid element is a group that quantity is maximum and research is the most deep in the bacteriocin, mainly comprises enterococcin (enterocin), pediocin small molecules bacteriocins such as (pediocin).This bacterioid element not only suppresses some corrupt milk-acid bacteria, rope silk bacterium (Brochotrix spp.), bacillus fusiformis (Clostridium spp.), genus bacillus (Bacillus spp.), staphylococcus (Staphylococcus spp.) etc., and the foodborne bacterial pathogens list is increased listeria spp (Listeria monocylogenes) had strong inhibitory effects is arranged, have the potential using value of better controlled food spoilage bacterium and pathogenic bacteria.In addition, compare with other bacteriocin, the bacteriostatic activity of II a bacterioid element is strong and have good physicochemical property, is the bacteriocin that is hopeful to be applied to multiple industrial use most of generally acknowledging at present.
In recent years, make up the lactobacillus food grade heterologous expression system, probe into some IIa bacterioid plain genes that the huge applications prospect is arranged in milk-acid bacteria the clone and express the research focus that has become the bacteriocin lab molecular genetics.Yet traditional lactic acid bacteria expression vectors mostly with antibiotics resistance genes such as erythromycin or paraxin as selection markers, though this keeps certain selective pressure during for genetic manipulation and effectively selects transformant, but render to antibiotics resistance gene in the environment or the humans and animals body in, because the hidden danger that exists resistance factor to shift, may bring the serious consequence of biological safety, so consider that from the food safety angle containing the antibiotics resistance gene microorganisms marked can not be applied in the foodstuff production.And in order to prevent to use the caused harm of antibiotics resistance mark, the most effective way is to use the food grade selection markers to human body safety to substitute the antibiotics resistance mark with structuring food prods level acceptor and carrier system.
Summary of the invention
An object of the present invention is to provide a kind of genetic engineering bacterium method for preparing bacteriocinogeny.
The genetic engineering bacterium method of preparation bacteriocinogeny provided by the present invention comprises the steps: the encoding gene importing host bacterium of bacteriocin is obtained the bacterium of recombinating; From described reorganization bacterium, obtain to express the genetic engineering bacterium of described bacteriocin; The aminoacid sequence of described bacteriocin is shown in sequence in the sequence table 3.
The encoding gene of described bacteriocin is following A) or B) shown in:
A) encoding gene of described bacteriocin is following 1) or 2) or 3) in arbitrary described dna molecular:
1) dna molecular shown in the sequence in the sequence table 1;
2) under stringent condition with 1) dna molecule hybridize and have the dna molecular of identical function;
3) with 1) or 2) dna molecular have 90% above homology and have the dna molecular of identical function;
B) encoding gene of described bacteriocin is following 4) or 5) or 6) in arbitrary described dna molecular:
4) dna molecular shown in the sequence in the sequence table 2;
5) under stringent condition with 4) dna molecule hybridize and have the dna molecular of identical function;
6) with 4) or 5) dna molecular have 90% above homology and have the dna molecular of identical function.
Described host bacterium is Lactococcus lactis (Lactococcus lactis).
The encoding gene of described bacteriocin imports the host bacterium by recombinant expression vector.
Described recombinant expression vector is the recombinant expression vector that obtains between the BamHI of the encoding gene insertion expression vector pLEB590 of described bacteriocin and the XhoI restriction enzyme site.
The genetic engineering bacterium that is prepared by described method also belongs to protection scope of the present invention.
The application of described genetic engineering bacterium in the preparation bacteriocin also belongs to protection scope of the present invention.
Another object of the present invention provides a kind of method of a preparation bacteriocin.
The method of a preparation provided by the present invention bacteriocin, the described genetic engineering bacterium that comprises the steps: to ferment promptly obtains bacteriocin.
The substratum of described fermentation is the GM17 liquid nutrient medium;
The temperature of described fermentation is 30 ℃-37 ℃ or 37 ℃ or 30 ℃ or 34 ℃;
The time of described fermentation is 18 hours-32 hours or 18 hours-24 hours;
The volume percent of the inoculum size of described fermentation is 1%.
Described method comprises that also from fermented liquid supernatant separation and purification obtains the step of described bacteriocin after described fermentation; The method of described separation and purification may further comprise the steps:
(a) fermented liquid supernatant is saltoutd with 80% saturation ratio ammonium sulfate precipitation, collecting precipitation obtains the bacteriocin crude extract;
(b) the bacteriocin crude extract that step (a) is obtained carries out gel permeation chromatography, carries out wash-out with the 0.02mol/L phosphoric acid buffer, collects the elutriant with bacteriostatic activity, promptly obtains being first pure bacteriocin sample; The model of the gel column that adopts in the described gel permeation chromatography is Sephadex G-10;
(c) elutriant that step (b) is collected carries out cation exchange column chromatography, carries out linear elution with the 0.02mol/L acetate buffer solution that contains 0M-1M NaCl, collects the elutriant with bacteriostatic activity, promptly obtains bacteriocin; The model of the cationic exchange coloum that adopts in the described cation exchange column chromatography is SP Sepharose Fast Flow; The concentration of NaCl is at 40min-60min or 40min or 50min or rise to 1M by the 0M linearity in 60min minute in the described linear gradient elution.
The genetically engineered Lactococcus lactis of production provided by the present invention II a bacterioid element can be realized overexpression, continuous production and the rapid detection of II a bacterioid element.Utilizing this genetically engineered Lactococcus lactis, can extract from this genetically engineered Lactococcus lactis fermented liquid of every 1000mL and be purified to 5.66mg, is 5.12 * 10 than vigor
5The bacteriocin enterocin P of AU/mg.Simultaneously, this reorganization bacterium is screened with selective pressure material nisin, nisin is generally regarded as safe antiseptics for natural food, and the nisI gene source is in milk-acid bacteria self, and this makes the nisI gene is more had superiority as the lactobacillus food grade selection markers.
Description of drawings
Fig. 1 is the building process figure of recombinant expression plasmid pLEB590-sEntP1 and pLEB590-sEntP2.
Fig. 2 is the evaluation electrophorogram of recombinant expression plasmid.
Fig. 3 is the growth curve chart of reorganization Lactococcus lactis.
Fig. 4 is that the Tricine SDS-PAGE and the bacteriostatic activity of bacteriocin behind the purifying detects figure.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
One, produces the structure of the genetically engineered Lactococcus lactis of bacteriocin
1, the clone of Enterocin P encoding gene
1) design of primers is with synthetic
Nucleotide sequence (Original accession number:AF005726.1) (aminoacid sequence of enterocin P is shown in sequence in the sequence table 3) according to known coded bacteriocin enterocinP among the multiple clone site (MCS) of plasmid pLEB590 and the GenBank, on Primer 5.0, design primer and insert two restriction enzyme sites (BamHI and XhoI), see Table 1.
Table 1PCR amplification enterocin P encoding gene primer and product
A: indicating the underscore sequence partly is the restriction enzyme site of BamHI and XhoI.
2) pcr amplification
With faecium (Enterococcus faecium) LM-2 genomic dna is template, adopts the PCR reaction kit to carry out the amplified reaction of target gene fragment.The PCR reaction adopts 50 μ L systems to carry out, and concrete reaction system is as follows:
Table 2PCR reaction system
Behind the instantaneous centrifugal and mixing said mixture, increase by following reaction conditions: 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 45s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations, 72 ℃ are extended 10min.Getting PCR product 4 μ L, is dna molecular marker with DL 2000, and monobromomethane Finland is dyestuff, and 1% agarose gel electrophoresis detects the PCR product.The size of the PCR product that obtains after testing is identical with the plan theoretical size of amplified fragments (with reference to table 1).To remain the PCR product and directly reclaim and purifying, obtain purpose fragment sEntP1 and purpose fragment sEntP2 by using DNA to reclaim test kit.
Above-mentioned faecium (Enterococcus faecium) LM-2 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 05 09th, 2011, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.4832.
2, the structure of Enterocin P recombinant expression vector
1) carrier and the segmental enzyme of insertion are cut and are reclaimed
Respectively with above-mentioned purpose fragment sEntP1, the purpose fragment sEntP2 that obtains and expression vector pLEB590 (available from Valio company limited) BamHI and XhoI double digestion; Using DNA to reclaim test kit reclaims and purifying DNA fragment, detect enzyme by agarose gel electrophoresis and cut the result, and purpose fragment sEntP1 and purpose fragment sEntP2 after enzyme cut check order, and sequencing result is: the nucleotide sequence of purpose fragment sEntP1 is shown in sequence in the sequence table 1; The nucleotide sequence of purpose fragment sEntP2 is shown in sequence in the sequence table 2.
Expression vector pLEB590 comprises pSH 7l replicon, constitutive promoter P
45, and be marker gene with nisin immunogene nisI.
2) connect
To be connected with expression vector pLEB590 fragment with the purpose fragment sEntP1 of XhoI double digestion and purifying recovery through BamHI; Simultaneously, will be connected with expression vector pLEB590 fragment with the purpose fragment sEntP2 of XhoI double digestion and purifying recovery through BamHI; The construction of recombinant plasmid process as shown in Figure 1.10 μ L reorganization linked system is as shown in table 3 below:
Table 3 ligation system
Behind instantaneous centrifugal mixing, 4 ℃ of connections of spending the night obtain recombinant plasmid pLEB590-sEntP1 and recombinant plasmid pLEB590-sEntP2 with the solution in the above reaction system.
3) evaluation of recombinant expression vector
Extract recombinant plasmid pLEB590-sEntP1 and recombinant plasmid pLEB590-sEntP2 respectively, adopt pcr amplification, double digestion reaction and sequencing analysis to identify recombinant plasmid.The reaction system of pcr amplification, reaction conditions and the primer and step 2) identical.The double digestion reaction system is as follows:
Table 4 endonuclease reaction system
Behind 37 ℃ of reaction 3h, detect with 1% agarose gel electrophoresis, detected result is shown in (b) among (a) and Fig. 2 among Fig. 2; (a) is the qualification result of recombinant plasmid pLEB590-sEntP1 among Fig. 2, and wherein swimming lane M1 is 1Kb DNAladder, from top to bottom molecular weight be followed successively by 1000,8000,7000,6000,5000,4000,3000,2000,1000,500bp; Swimming lane M2 is Low range DNA ladder, from top to bottom molecular weight successively 2000,1500,1000,800,700,600,500,400,300,200,100bp; Swimming lane 1 is cut product for the not enzyme of recombinant plasmid pLEB590-sEntP1; Swimming lane 2 is cut product for the not enzyme of plasmid pLEB590; Swimming lane 3 is a recombinant plasmid pLEB590-sEntP1 double digestion product; Swimming lane 4 is cut product for plasmid pLEB590 enzyme; Swimming lane 5 is the PCR product of recombinant plasmid pLEB590-sEntP1.(b) is the qualification result of recombinant plasmid pLEB590-sEntP2 among Fig. 2, and wherein swimming lane M1 is identical with (a) among Fig. 2; Swimming lane M2 is identical with (a) among Fig. 2; Swimming lane 1 is cut product for the not enzyme of recombinant plasmid pLEB590-sEntP2; Swimming lane 2 is cut product for the not enzyme of plasmid pLEB590; Swimming lane 3 is a recombinant plasmid pLEB590-sEntP2 double digestion product; Swimming lane 4 is cut product for plasmid pLEB590 enzyme; Swimming lane 5 is the PCR product of recombinant plasmid pLEB590-sEntP2.(a) as seen comprises 311bp purpose fragment sEntP1 among the recombinant plasmid pLEB590-sEntP1 from Fig. 2; (b) as seen comprises 529bp purpose fragment sEntP2 among the recombinant plasmid pLEB590-sEntP2 from Fig. 2.
To cut through PCR and enzyme and identify that correct recombinant plasmid send order-checking company to check order, use DNAMAN (Version 5.0) software analysis sequencing result.
With original sequence alignment among the sequencing result of recombinant plasmid and the Genebank, the purpose fragment sEntP1 and sEntP2 and original series of cloning homology on encoder block be 100%.Above presentation of results recombinant plasmid pLEB590-sEntP1 and recombinant plasmid pLEB590-sEntP2 successfully make up.
4) preparation of Lactococcus lactis MG1614 competent cell
The single bacterium colony of Lactococcus lactis of picking (Lactococcus lactis) MG1614 (available from Valio company limited) from the fresh GM17 agar plate is inoculated in GM17 liquid nutrient medium (Tryptones 5g/L, soy peptone 5g/L, extractum carnis 2.5g/L, yeast extract paste 5g/L, xitix 0.5g/L, MgSO
47H
2O 0.25g/L, Sodium phosphate dibasic 10g/L and lactose 5g/L) in, 30 ℃ leave standstill overnight incubation; With above-mentioned culture by volume per-cent be that 1% inoculum size is inoculated in the SGM17 liquid nutrient medium (the GM17 substratum that contains 2.0% glycine and 0.5mol/L sucrose) that contains 3.0% glycine and preheating, 30 ℃ leave standstill and are cultured to OD600 and reach 0.7; Take out culture, place cooled on ice 30min.Slowly evenly shake during this time, to guarantee that culture fully cools off; Above-mentioned nutrient solution is transferred in the centrifuge tube of precooling, 4 ℃, behind the centrifugal 10min of 6000rpm, abandon supernatant liquor, electricity with the precooling of 1/2 original volume transforms buffer solution for cleaning cell 2 times, at last with cell suspension in precooling electricity conversion damping fluid with 1/100 nutrient solution volume; Cell suspension is sub-packed in the 1.5mL Eppendorf tube by 100 μ L, is stored in-80 ℃, standby.
5) electricity transforms Lactococcus lactis MG1614 competent cell
Draw the competent cell suspension of 50 μ L prepared fresh and go in the ice-cold micro-aseptic centrifuge tube of 0.5mL, ice is educated 5min.Add 2 μ L recombinant plasmid pLEB590-sEntP2 to be transformed, obtain mixed solution, ice is educated 5min; Mixed solution is added ice-cold 2mm electric shock transform in the cup, touch the electric shock cup and be positioned at electric shock cup bottom, and must not produce foam to guarantee mixed solution; Dry electric shock tank outer water of condensation and fog; The cup that will shock by electricity is put into the electric shock instrument.Regulate the electric shock instrument, making electricimpulse is 25 μ F, voltage 2.5kV, resistance 200 Ω, carries out electricimpulse; After electricimpulse finishes, from electric shock tank, take out the electric shock cup as early as possible, add the ice-cold SGM17MC regeneration liquid nutrient medium of 1mL; Be transferred to after being mixed in the 1.5mL Eppendorf tube, 30 ℃, leave standstill incubation 2h; Get the culture that 100-200 μ L cultivates 2h, coat in the GM17 substratum that contains 200IU/mL nisin (nisin), cultivate down for 30 ℃, can occur bacterium colony in the 48h, promptly obtain containing the recombination lactic acid galactococcus of recombinant plasmid pLEB590-sEntP2.Contain recombinant plasmid pLEB590-sEntP1 recombination lactic acid galactococcus MG1614 and contain the recombination lactic acid galactococcus MG1614 of recombinant plasmid pLEB590-sEntP2, so can in containing the substratum of nisin, grow owing to comprise nisin immunogene nisI.
With the recombination lactic acid galactococcus that above-mentioned same method obtains containing the recombination lactic acid galactococcus of recombinant plasmid pLEB590-sEntP1 and contains plasmid pLEB590, the recombination lactic acid galactococcus that will contain plasmid pLEB590 is as changeing empty carrier contrast reorganization recombination lactic acid galactococcus; Establishing unconverted Lactococcus lactis (Lactococcus lactis) MG1614 simultaneously is the wild-type contrast.
Two, produce the application of the genetically engineered Lactococcus lactis of bacteriocin
Method I
1, recombinant expressed host's growth and bacteriostatic activity analysis
To contain the recombination lactic acid galactococcus of recombinant plasmid pLEB590-sEntP1, the recombination lactic acid galactococcus cell that contains recombinant plasmid pLEB590-sEntP2 and commentaries on classics empty carrier contrast recombination lactic acid galactococcus cell is 1% (about 10 with volume percent respectively
5CFU/mL) inoculum size is inoculated in the GM17 liquid nutrient medium, leaves standstill cultivation under 37 ℃, obtains fermented liquid; (0-48h) gets fermented liquid at different time, surveys OD
600With the inhibitory potency value, draw strain growth and bacteriostatic activity curve, and the growth characteristics of more different recombination lactic acid galactococcus transformant cells.
The method of measuring the inhibitory potency value is as follows:
1. prepare double-deck agar plate: (Tryptones 17g/L, soya peptone 3g/L, yeast soak powder 6g/L, NaCl 5g/L, K at first to get 6mL TSYE solid medium
2HPO
42.5g/L, glucose 2.5g/L and agar 13g/L) be tiled in the plate of 9cm diameter, place to make agar layer form uniform thickness on the level table, (Tryptones 17g/L, soya peptone 3g/L, yeast soak powder 6g/L, NaCl 5g/L, K to be taken at the TSYE liquid nutrient medium then
2HPO
42.5g/L fresh culture and be diluted to 5.0 * 10 and glucose 2.5g/L)
7Monocyte hyperplasia listeria spp (L.monocytogenes) NICPBP 54002 (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) (indicator) the 100 μ L of CFU/mL add in the warm 10mL TSYE agar test tube, fall in plate behind the mixing gently, place to keep the substratum consistency of thickness on the level table.
2. prepare testing sample: the fermented liquid of getting above-mentioned different time (0-48h) is at 8000rpm, and 4 ℃ of low-temperature centrifugation 10min remove bacterial sediment, and the collection fermented supernatant fluid is a testing sample, deposits in 4 ℃ of refrigerators standby.
3. doubling dilution is measured inhibitory potency: sample is carried out doubling dilution with the PBS phosphoric acid buffer of pH 6.5, the 100 μ L that take a sample carry out bacteriostatic test, the high dilution that will not observe that inhibition zone occurs is defined as a unit of activity (1AU), and 10 times of its inverse promptly is the valence value (AU/mL) of bacteriocin sample.
The method of bacteriostatic test is as follows: open plate lid and place the sterile air 30min that dries in the air, and with aseptic nipper with Oxford cup (6mm * 7.8mm * 10mm, available from the U.S. happy food machinery in Xinxiang, Henan factory available from) be positioned on the flat board gently, the middle certain distance that keeps, to wherein adding testing sample separately, under keeping horizontal state, flat board is put in spread in 4 ℃ of refrigerators gently and spends the night, place 37 ℃ of cultivations down then, occur until inhibition zone.
The OD of thalline
600The results are shown in Figure 3 with the inhibitory potency value, the somatic cells growth OD from figure
600The value curve as seen, contain the recombination lactic acid galactococcus cell of recombinant plasmid pLEB590-sEntP1 and contain recombinant plasmid pLEB590-sEntP2 recombination lactic acid galactococcus cell and all can have the growth curve of cell density much at one 37 ℃ of growths down with commentaries on classics empty carrier contrast recombination lactic acid galactococcus cell; Wild-type contrast Lactococcus lactis cell growth OD
600The value curve does not have significant difference with commentaries on classics empty carrier contrast recombination lactic acid galactococcus cell.This shows that the recombinant plasmid transformed of carrying purpose fragment sEntP1 and purpose fragment sEntP2 enters after the Lactococcus lactis MG1614, does not have a negative impact to its growth.And the analytical results of inhibitory potency shows: change the empty carrier control strain and do not show any bacteriostatic activity in whole growth process, and the recombination lactic acid galactococcus that contains recombinant plasmid pLEB590-sEntP1 begins to produce bacteriocin with the recombination lactic acid galactococcus that contains recombinant plasmid pLEB590-sEntP2 from logarithmic growth mid-term (about 6h), reach highest level stablizing mid-term (about 18-36h), this shows that bacteriocin enterocin P can obtain expression by food grade vector pLEB590 in Lactococcus lactis.In addition, the recombination lactic acid galactococcus institute bacteriocinogeny bacteriostatic activity that the result also finds to contain recombinant plasmid pLEB590-sEntP2 is apparently higher than the recombination lactic acid galactococcus that contains reorganization recombinant plasmid pLEB590-sEntP1, and this shows that the coexpression of enterocin P immunogene can obviously strengthen the secreting, expressing level of this bacteriocin in the Lactococcus lactis heterologous expression system.Selection is next step test strain with the recombination lactic acid galactococcus that contains recombinant plasmid pLEB590-sEntP2.
2, the extraction purifying of bacteriocin
The extraction purifying of bacteriocin extracts purifying and adopts three step purifying procedures: comprise that ammonium sulfate precipitation concentrates, gel permeation chromatography and cation exchange resin layer analyse, be indicator with monocyte hyperplasia listeria spp (L.monocytogenes) NICPBP 54002 in the purge process, and adopt agar diffusion method that per step purification of samples bacteriostatic activity is analyzed.Concrete grammar is as follows:
Adopt 80% saturation ratio ammonium sulfate precipitation to contain slightly carrying of bacteriocin in 18 hours the fermented supernatant fluid of fermentation of recombination lactic acid galactococcus of recombinant plasmid pLEB590-sEntP2, collecting precipitation also redissolves it in the 0.02mol/L of original volume 1/10 phosphoric acid buffer (pH 6.0), and the preparation gained is the bacteriocin crude extract.The result can obtain 100mL from the 1000mL fermented supernatant fluid, than vigor 2.17 * 10
4The bacteriocin crude extract of AU/mg; Then carry out purifying by Sephadex G-10 gel permeation chromatography, the last sample 1mL bacteriocin product that slightly get sample adopt pH 6.0,0.02mol/L phosphoric acid buffer wash-out, and flow velocity is 0.5mL/min, and the OD of elutriant is respectively managed in monitoring simultaneously
280Value.According to OD
280The situation of value figure Wave crest and wave trough is carried out bacteriostatic activity after the elutriant at all peaks merged respectively and is detected.Collect activated sample as first pure bacteriocin sample, 4 ℃ of freezer storages are standby.The result can obtain 150mL, than vigor 4.91 * 10
5The first pure bacteriocin sample of AU/mg; At last, using SP Sepharose Fast Flow cation exchange resin layer to analyse is further purified, last sample 1.5mL is the pure bacteriocin sample just, to contain the 0.02mol/L acetate buffer solution linear gradient elution of 0-1M NaCl, pH 6.5, the time of linear gradient elution is 40min, and the concentration of NaCl rises to 1M by the 0M linearity in the described linear gradient elution in 40min), flow velocity is 0.5mL/min, detect the ultraviolet absorption value of elutriant simultaneously at the 280nm place, and according to OD
280The situation of value figure Wave crest and wave trough is carried out bacteriostatic activity after the elutriant at all peaks merged respectively and is detected.Collect activated sample as the purification of bacterial element, finally obtain 50mL, than the plain sample (table 5) of the purification of bacterial of vigor 5.12 * 105AU/mg.
The purifying of table 5 recombinant bacteria element
As can be seen from Table 5, by above three step purifying procedures (ammonium sulfate precipitation, gel filtration chromatography and cation exchange resin layer are analysed), the fermented liquid that every 1000mL contains the recombination lactic acid galactococcus of recombinant plasmid pLEB590-sEntP2 can finally obtain 5.66mg, is 5.12 * 10 than vigor
5The bacteriocin of AU/mg.
Total protein: employing BCA determination of protein concentration test kit (available from green skies biotechnology research institute, product article No.: P0012) total protein content in each sample is measured;
Total activity is defined as: the total antibacterial unit of activity number that the bacteriocin sample is had;
Be defined as than vigor: every milligram of antibacterial unit of activity number that bacteriocin albumen is had;
Total activity (AU)=bacteriocin sample valence value (AU/mL) * population of samples amasss (mL);
Than vigor (AU/mg)=bacteriocin sample total activity (AU)/sample total protein content (mg);
Behind purifying multiple=purifying the bacteriocin sample than the preceding bacteriocin sample of vigor (AU/mg)/purifying than vigor (AU/mg);
Bacteriocin sample total activity (AU) * 100% before bacteriocin sample total activity (AU)/purifying behind the rate of recovery (%)=purifying.
3, the activity of bacteriocin detects behind the purifying
Bacteriocin behind the Tricine SDS-PAGE electrophoresis detection purifying: get the plain sample of 14 μ L purification of bacterial and mix with 7 μ L sample-loading buffers, last sample is preceding at 65 ℃ of 15min that precook.Same two glue handling carry out electrophoresis simultaneously, after electrophoresis finishes, wherein a glue dyes with the Xylene Brilliant Cyanine G dye liquor, another piece glue is with 25% Virahol (volume percent), 10% acetate (volume percent) immobilized gel 20min, use phosphate buffered saline buffer (5mmol/L, pH 6.5) flushing 1h to be used to detect bacteriostatic activity again.
Simultaneously, the recombination lactic acid galactococcus cell that will contain recombinant plasmid pLEB590-sEntP2 is 1% (about 10 with volume percent respectively
5CFU/mL) inoculum size is inoculated in the GM17 liquid nutrient medium, leave standstill under 37 ℃ and cultivated 18 hours, then with this fermented liquid at 8000rpm, 4 ℃ of low-temperature centrifugation 10min, remove bacterial sediment, the collection fermented supernatant fluid is a testing sample, deposits in 4 ℃ of refrigerators standbyly, uses the method identical with bacteriocin behind the above-mentioned detection purifying to detect bacteriocin in the fermented supernatant fluid.
Bacteriostatic activity detects: with the indicator cell inoculation in logarithmic growth mid-term of fresh culture in 10mL, contain in the TSYE substratum of 1% low electric osmose agarose and 0.02% tween 20, and make final indicator concentration be about 4.0 * 10
6CFU/mL pours flat board into behind the mixing rapidly, and running gel to be detected is placed on it, remove running gel behind 37 ℃ of cultivation 3h, aseptic nutrient agar with 10mL covers (contain the double nutritive ingredient of TSYE, and add 0.8% low electric osmose agarose), 37 ℃ of appearance of cultivating and observing inhibition zone.
Contain recombination lactic acid galactococcus fermented supernatant fluid component and the Tricine SDS-PAGE electrophoresis of purifying gained bacteriocin sample and the result of usefulness coomassie brilliant blue staining of recombinant plasmid pLEB590-sEntP2, (swimming lane 1 is a standard molecular weight albumen shown in (a) among Fig. 4; Swimming lane 2 is a fermented supernatant fluid; Swimming lane 4 is a bacteriocin behind the purifying), (swimming lane 4 is a fermented supernatant fluid to the bacteriostatic activity detected result shown in (b) among Fig. 4; Swimming lane 5 is a bacteriocin behind the purifying).As seen from the figure, the activated protein band about all visible 4500Da in fermented supernatant fluid and the purification of samples.Though because expression amount is lower, Tricine SDS-PAGE electrophoresis purpose band is dim, but electrophoresis sepharose diffusion test still can detect tangible bacteriostatic activity, and it is feasible effective that this explanation utilizes food grade expression vector pLEB590 allos secreting, expressing biological activity enterocin P.
Method II
1, recombinant expressed host's growth and bacteriostatic activity analysis
Except that leavening temperature was 30 ℃, all the other methods were all identical with method I.
Result and method I do not have significant difference.
2, the extraction purifying of bacteriocin
The time of decationize exchange resin chromatography neutral line gradient elution is beyond the 50min, and all the other methods are identical with method I.
Result and method I do not have significant difference.
Method III
1, recombinant expressed host's growth and bacteriostatic activity analysis
Except that leavening temperature was 34 ℃, all the other methods were all identical with method I.
Result and method I do not have significant difference.
2, the extraction purifying of bacteriocin
The time of decationize exchange resin chromatography neutral line gradient elution is beyond the 60min, and all the other methods are identical with method I.
Result and method I do not have significant difference.
Claims (10)
1. a genetic engineering bacterium method for preparing bacteriocinogeny comprises the steps: the encoding gene importing host bacterium of bacteriocin is obtained the bacterium of recombinating; From described reorganization bacterium, obtain to express the genetic engineering bacterium of described bacteriocin; The aminoacid sequence of described bacteriocin is shown in sequence in the sequence table 3.
2. method according to claim 1 is characterized in that:
The encoding gene of described bacteriocin is following A) or B) shown in:
A) encoding gene of described bacteriocin is following 1) or 2) or 3) in arbitrary described dna molecular:
1) dna molecular shown in the sequence in the sequence table 1;
2) under stringent condition with 1) dna molecule hybridize and have the dna molecular of identical function;
3) with 1) or 2) dna molecular have 90% above homology and have the dna molecular of identical function;
B) encoding gene of described bacteriocin is following 4) or 5) or 6) in arbitrary described dna molecular:
4) dna molecular shown in the sequence in the sequence table 2;
5) under stringent condition with 4) dna molecule hybridize and have the dna molecular of identical function;
6) with 4) or 5) dna molecular have 90% above homology and have the dna molecular of identical function.
3. method according to claim 1 and 2 is characterized in that:
Described host bacterium is Lactococcus lactis (Lactococcus lactis).
4. according to arbitrary described method among the claim 1-3, it is characterized in that:
The encoding gene of described bacteriocin imports the host bacterium by recombinant expression vector.
5. according to arbitrary described method among the claim 1-4, it is characterized in that:
The recombinant expression vector that described recombinant expression vector obtains for the multiple clone site of the encoding gene of described bacteriocin being inserted expression vector pLEB590.
6. the genetic engineering bacterium for preparing by arbitrary described method among the claim 1-5.
7. the application of the described genetic engineering bacterium of claim 6 in the preparation bacteriocin.
8. method for preparing bacteriocin, the described genetic engineering bacterium of claim 6 that comprises the steps: to ferment promptly obtains bacteriocin.
9. method according to claim 8 is characterized in that:
The temperature of described fermentation is 30 ℃-37 ℃ or 37 ℃ or 30 ℃ or 34 ℃;
The time of described fermentation is 18 hours-32 hours or 18 hours-24 hours;
The volume percent of the inoculum size of described fermentation is 1%.
10. it is characterized in that according to Claim 8 or 9 described methods:
Described method comprises that also from fermented liquid supernatant separation and purification obtains the step of described bacteriocin after described fermentation; The method of described separation and purification may further comprise the steps:
(a) fermented liquid supernatant is saltoutd with 80% saturation ratio ammonium sulfate precipitation, collecting precipitation obtains the bacteriocin crude extract;
(b) the bacteriocin crude extract that step (a) is obtained carries out gel permeation chromatography, carries out wash-out with the 0.02mol/L phosphoric acid buffer, collects the elutriant with bacteriostatic activity, promptly obtains being first pure bacteriocin sample; The model of the gel column that adopts in the described gel permeation chromatography is Sephadex G-10;
(c) elutriant that step (b) is collected carries out cation exchange column chromatography, carries out linear elution with the 0.02mol/L acetate buffer solution that contains 0M-1M NaCl, collects the elutriant with bacteriostatic activity, promptly obtains bacteriocin; The model of the cationic exchange coloum that adopts in the described cation exchange column chromatography is SP Sepharose Fast Flow; The concentration of NaCl rises to 1M by the 0M linearity in the described linear gradient elution in 40min-60min or 40min or 50min or 60min.
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| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
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| CN108776222A (en) * | 2018-06-14 | 2018-11-09 | 吉林省农业科学院 | Gongzhuling mycin bacteriostatic activity detection method and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531562A (en) * | 2014-12-09 | 2015-04-22 | 北京农学院 | Preparation method of (Lactobacillus planetarium subsp. plantarum)Zhang-LL and its listeria monocytogene-resistant bacteriocin |
| CN104531562B (en) * | 2014-12-09 | 2018-09-14 | 北京农学院 | A kind of preparation method of lactobacillus plantarum plant subspecies and its anti-Listeria monocytogenes bacteriocin |
| CN104845925A (en) * | 2015-05-22 | 2015-08-19 | 安徽民祯生物工程有限公司 | Lactococcus lactis and method for simultaneously producing nisin and pediocin by using lactococcus lactis |
| CN108776222A (en) * | 2018-06-14 | 2018-11-09 | 吉林省农业科学院 | Gongzhuling mycin bacteriostatic activity detection method and application |
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